Human Molecular Genetics, 2000, Vol. 9, No. 5 821-828
© 2000 Oxford University Press
The mouse neurological mutant flailer expresses a novel hybrid gene derived by exon shuffling between Gnb5 and Myo5a
1Department of Human Genetics, 4708 Medical Science II, University of Michigan, Ann Arbor, MI 48109-0618, USA, 2Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA, 3Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA and 4Department of Medicine, University of California, San Diego, CA 92093, USA
Received 9 December 1999; Revised and Accepted 12 January 2000.
DDBJ/EMBL/GenBank accession nos AF174490, AF174491 and AF176041.
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ABSTRACT |
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Exon shuffling is thought to be an important mechanism for evolution of new genes. Here we show that the mouse neurological mutation flailer (flr) expresses a novel gene that combines the promoter and first two exons of guanine nucleotide binding protein beta 5 (Gnb5) with the C-terminal exons of the closely linked Myosin 5A (MyoVA) gene (Myo5a). The flailer protein, which is expressed predominantly in brain, contains the N-terminal 83 amino acids of Gnb5 fused in-frame with the C-terminal 711 amino acids of MyoVA, including the globular tail domain that binds organelles for intracellular transport. Biochemical and genetic studies indicate that the flailer protein competes with wild-type MyoVA in vivo, preventing the localization of smooth endoplasmic reticulum vesicles in the dendritic spines of cerebellar Purkinje cells. The flailer protein thus has a dominant-negative mechanism of action with a recessive mode of inheritance due to the dependence of competitive binding on the ratio between mutant and wild-type proteins. The chromosomal arrangement of Myo5a upstream of Gnb5 is consistent with non-homologous recombination as the mutational mechanism. To our knowledge, flailer is the first example of a mammalian mutation caused by germ line exon shuffling between unrelated genes.
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INTRODUCTION |
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Myosin 5A (MyoVA) is a 1853 residue unconventional myosin heavy chain with major expression in brain and skin that functions as an actin-based molecular motor (1,2). The MyoVA gene (Myo5a) is located on mouse chromosome 9, and many ‘dilute’ alleles have been generated at this locus in mutagenesis experiments (3,4). Myo5a null animals have a lightened coat color due to defects in melanosome transport (3,5) and die 14–21 days after birth from a neurological defect associated with defects in smooth endoplasmic reticulum (SER) localization in cerebellar Purkinje cells (6,7). Myo5a is also tightly bound to vesicles containing synaptic vesicle markers (8,9). The MYO5A mutation in a human patient with Griscelli syndrome truncates the protein at codon 779, resulting in pigmentation dilution and a neurological disorder with marked delay in motor development, hypotonia and mental retardation (10).
We describe a new spontaneous mouse mutant that expresses a hybrid protein derived from Myo5a and the guanine nucleotide binding protein beta 5 gene (Gnb5), the neuronal ß subunit of the heterotrimeric G-protein receptor that couples cell surface receptors with intracellular second messenger systems. The protein structure of the ß subunit has been determined (11,12) but mutant alleles of Gnb5 have not previously been described.
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RESULTS |
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Genetic mapping and neurological phenotype of flailer mice
In an attempt to positionally clone the neurological mutation tumbler (tb), we found that one allele, tumbler-2J (tb2J), does not map to the tb locus on mouse chromosome 1. Instead this autosomal recessive mutation, re-named flailer (flr), mapped to a 0.7 ± 0.4 cM interval on chromosome 9 (Fig. 1). The flailer mutation arose in 1971 on the C57BL/10J strain (H. Sweet, personal communication) and is maintained in our laboratory as the recombinant congenic line BcC-flr. Homozygous flr mice display neurological abnormalities by 14 days of age, including frequent falling and convulsive limb movements (leg flailing) when attempting to right themselves. The frequency of convulsions increases between 2 and 4 weeks of age. Opisthotonus (arching of the head and tail) can be stimulated during this period by handling. Swimming ability and pigmentation are normal. When placed on an accelerating Rotorod rotating cylinder, wild-type mice maintain themselves in an upright position for 3–4 min whereas flailer homozygotes fall off within 0.5–1 min. Motor coordination improves with age, but adult flr mice continue to exhibit mild ataxia and cannot balance on a narrow beam. Fertility and life span are normal in the BcC-flr line.
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The neurological phenotype of flailer homozygotes at 3 weeks of age closely resembles that of homozygotes for null alleles of Myo5a, although the Myo5a null mice do not survive beyond weaning (4). Since Myo5a was previously mapped to central chromosome 9, we examined its segregation in the flailer mapping cross. No recombinants were observed between flailer and Myo5a, demonstrating close linkage (Fig. 1).
Defect in localization of SER in cerebellar Purkinje cells
Abnormal localization of SER vesicles was observed by transmission electron microscopy of cerebellum from 20-day-old affected homozygous flr mice. SER is clearly visible in dendritic spines adjacent to regions of postsynaptic density between parallel fibers and Purkinje cells of wild-type controls (Fig. 2a). However, in flailer Purkinje cells the dendritic spines are devoid of SER (Fig. 2b). The similarity of this cellular defect to that reported for Myo5a null mice (7) indicated further that Myo5a function might be affected by the flailer mutation.
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Normal and hybrid Myo5a transcripts in flailer brain
Northern analysis using a Myo5a head region probe detected the three normal Myo5a transcripts of 11, 7 and 6 kb in flailer brain (Fig. 3a, left). Complete RT–PCR sequencing of the 5.5 kb Myo5a open reading frame also failed to detect any difference between flr and wild-type transcripts (data not shown). However, when a Myo5a tail region probe was used for northern analysis, three abnormal transcripts of 8, 4 and 3 kb were detected in flailer brain (Fig. 3a, middle). The abundance of the abnormal transcripts is slightly higher than the wild-type Myo5a transcripts.
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Since the abnormal transcripts do not hybridize with a head region probe, and since each abnormal transcript is 3 kb shorter than one of the wild-type transcripts, we hypothesized that the three novel transcripts might be initiated from a common start site located ~3 kb downstream of a common start site for the three normal Myo5a transcripts. To test this model, we performed 5' RACE using Myo5a PCR primers M1R, M2R and M3R located 3.7–3.9 kb downstream of the 5' terminus of the 7 kb Myo5a transcript. A discrete 533 bp fragment was amplified from homozygous flr RNA but not from wild-type RNA. BLAST analysis of the sequence demonstrated that the 5' end of this RACE product was identical to Gnb5 cDNA nucleotides 6–278 (GenBank accession no. L34290) and the 3' end matched Myo5a cDNA nucleotides 3464–3723 (GenBank accession no. X57377) (Fig. 4a).
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RT–PCR amplification of brain RNA using Gnb5 forward and Myo5a reverse primers confirmed that Gnb5–Myo5a hybrid transcripts are expressed in flr mice (Fig. 4b, right). In addition, hybridization of northern blots with a Gnb5 cDNA probe demonstrated that the three abnormal transcripts detected with the Myo5a tail region probe also contain sequences from the 5' end of Gnb5 (Fig. 3a, right).
In addition to the hybrid transcript, wild-type Gnb5 transcripts were detected in flailer brain by RT–PCR (Fig. 4b, left) and by northern analysis (Fig. 3a, right). The abundance of the wild-type Gnb5 transcript is normal in flailer mice (Fig. 3a). Thus, flailer homozygotes express transcripts of three genes: wild-type Gnb5, wild-type Myo5a and the flailer hybrid gene.
Abnormal Myo5a protein in flailer brain
The hybrid transcript expressed in flr mice is predicted to encode a protein of 85 kDa with the first 83 amino acids derived from the N-terminus of Gnb5 and the last 711 amino acids from the C-terminus of MyoVA. To determine whether this hybrid protein is stably expressed, brain extracts from flr and wild-type mice were examined by western analysis. Using a MyoVA head region antibody, only the wild-type protein of 215 kDa was detected (Fig. 3b, left). However, with an antibody to the MyoVA tail region, an abnormal 85 kDa protein was detected in flr (Fig. 3b, middle). The predicted 85 kDa hybrid protein contains two of the three MyoVA dimerization domains and the distal MyoVA globular domain thought to be important for cargo binding, but is missing the actin-binding head region (Fig. 3c). Similar constructs are capable of competition for melanosome binding in transfected melanocytes (5).
Gene dosage and recessive inheritance
If competition by the hybrid flailer protein is responsible for the lack of accumulation of SER in the Purkinje cell dendritic spines shown in Figure 2, then the ratio of wild-type to mutant protein is expected to influence the expression of the phenotype. To determine whether the excess of wild-type protein is responsible for the lack of symptoms in flailer heterozygotes and recessive inheritance of the neurological disorder, we crossed flailer heterozygotes with mice heterozygous for a null allele of Myo5a. The compound heterozygous offspring exhibited the flailer phenotype (Table 1). This result supports the competitive binding hypothesis, and demonstrates that the in vivo ratio of flailer protein to wild-type protein is critical to generation of the neurological phenotype.
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Genetic and physical mapping of Gnb5
In order to elucidate the mutational mechanism that generated the hybrid flailer transcript, we mapped the mouse Gnb5 gene using the BSS backcross mapping panel (14). There were no recombinants between Gnb5 and Myo5a in this panel (0/92), demonstrating linkage of the two genes. YAC-based physical mapping showed that Gnb5 and Myo5a are both present on the 750 kb YAC clone YLE1294, with Myo5a upstream of Gnb5 (data not shown). Sequences derived from the human orthologs GNB5 and MYO5A are located on the same 220 kb BAC clone (GenBank/HTGS accession no. AC010674).
Identification of an intronic junction site in the hybrid flailer gene
To identify the genomic junction between Gnb5 and Myo5a sequences, the primers G1F and M5R flanking the cDNA junction site (Fig. 4a) were used to amplify flailer genomic DNA. A unique 3 kb flailer genomic fragment was amplified and sequenced (GenBank accession no. 176041). We also amplified and sequenced the corresponding 4 kb intron 2 from Gnb5 (GenBank accession no. AF174491) and the 2.3 kb intron ‘M’ from Myo5a (GenBank accession no. AF174490). The 3 kb flailer intron (Fig. 4c) contains 0.65 kb from the Gnb5 intron followed by nearly all of the 2.3 kb Myo5a intron. The breakpoint in the Gnb5 intron is located at position 35 of a partial (144 bp) B1 SINE element. There is little sequence homology between the two introns around the junction site (Fig. 5a), suggesting the rearrangement resulted from non-homologous recombination.
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It is interesting to note that each intron contains a copy of a BC1 ID element located ~100 bp downstream of the breakpoint site (Fig. 5b). If the breakpoint junctions in the two introns are aligned, the BC1 repeat sequences are oriented in the opposite direction and overlap by 31 bp. However, the 55% sequence identity (45/82 bp) between these two BC1 repeats may be too low to promote the recombination event that produced the flailer mutation. Comparison of the intron sequences using the programs Pipmaker (http://globin.cse.psu.edu/pipmaker/ ) and BLAST 2 Sequences (http://www.ncbi.nlm.nih.gov/BLAST/ ) did not identify additional regions of sequence similarity. Because Gnb5 intron 2 and Myo5a intron M are both phase 0 introns, the open reading frame is maintained in the hybrid gene.
Unique junction fragments containing both Myo5a and Gnb5 sequences were detected on Southern blots of flailer genomic DNA (Fig. 5c). All of the wild-type fragments of the Myo5a and Gnb5 genes can also be detected in flailer DNA using appropriate cDNA probes (data not shown).
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DISCUSSION |
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Chromosomal model for mutation by unequal crossover
The flailer mouse expresses an unusual mutant gene derived from two closely linked but unrelated parental genes. In addition to the mutant gene, the flailer chromosome retains wild-type copies of the parental genes Myo5a and Gnb5, as indicated by the presence of wild-type transcripts, introns and restriction fragments. Since Myo5a is closely linked and upstream of Gnb5 in the wild-type chromosome, misalignment and non-homologous recombination between Gnb5 and Myo5a could generate a chromosome containing the hybrid gene with retention of normal gene copies, as shown in Figure 6. This mutational mechanism requires a single event, whereas other models such as duplication and deletion require two or more events and therefore seem less likely.
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Many cases of unequal recombination between related gene copies in clustered multigene families have been observed in the human germ line, including recombination between globin genes and the generation of the hybrid red–green opsin gene that is a common cause of human color-blindness (15). However, the flailer mutation appears to be the first observed case of germ line recombination between unrelated parental genes.
The sequences around the junction site in the Myo5a and Gnb5 introns do not provide an obvious explanation for this rare event. The most similar sequences in the two introns are BC1 elements located ~100 bp from the junction sites in the Gnb5 and Myo5a introns. The Bc1 gene on chromosome 7 encodes a stable cytoplasmic RNA related to an alanine tRNA (16). There are ~5000 BC1 elements derived from the reverse transcribed cytoplasmic RNA in the mouse genome (17,18). The BC1 copies in the Gnb5 and Myo5a introns exhibit only 55% sequence identity, which may be too low to account for the misalignment of the introns.
Pathogenic mechanism of the myosin tail protein fragment in flailer mice
During the period between 2 and 4 weeks of age, the neurological phenotype of flailer mice is indistinguishable from that of Myo5a null mice homozygous for the dl20J allele. The loss of SER within dendritic spines of cerebellar Purkinje cells is a very specific cellular phenotype shared by both mutants. These similarities strongly suggest that interference with the function of wild-type MyoVA protein is the major mechanism of action of the new mutation. One mechanism by which the flailer hybrid protein could disrupt MyoVA function is through simple competition with the tail domain of the intact motor for tail binding sites. These would include presumed cargo such as the SER in dendritic spines, and possibly other proteins as well. Recent studies have shown that co-expression of tail domains of class V myosins can disrupt function of the intact motor. Transfection of cultured melanocytes with constructs encoding the same portion of MyoVA that is present in the flailer protein results in a ‘dilute’ phenotype in which pigment granules are transported but not retained in the peripheral dendrites of melanocytes and therefore clump around the nucleus (5). In Saccharomyces cerevisiae, co-expression of the globular tail domain of Myo2p, an essential class V myosin, completely disrupts its functions (19). In both instances, the expressed tail domain exhibits subcellular localization consistent with a cargo-competition mechanism.
Two other mechanisms may contribute to the dominant-negative effect of the flailer protein. The flailer protein includes portions of the dimerization domain (Fig. 3c) and may ‘poison’ the intact motor through heterodimerization. Since the processivity of myosin V is likely to require two heads (20), the heterodimer with a single head may be unable to function in transport or retention of SER. Competition for a dynein light chain that associates tightly with the tail domain of MyoVA is also possible (21,22). The lack of coat color dilution in flailer mice probably reflects the tissue specificity of the Gnb5 promoter, which is predominantly expressed in central nervous system (23,24) and is not expressed in skin (unpublished data).
Recessive inheritance and developmental recovery of the neurological phenotype
flailer heterozygotes carry two wild-type Myo5a alleles and one copy of the flailer hybrid gene. In heterozygous brain, the amount of flailer protein is insufficient to compete with the wild-type protein, and the mice are normal. In homozygotes with a 1:1 ratio of wild-type Myo5a and hybrid gene, effective competition is observed during the critical period of brain maturation between 2 and 4 weeks of age. Only one copy of the flailer gene is required to generate disease in animals heterozygous for a null allele of Myo5a, demonstrating the importance of the ratio of wild-type to mutant protein. The severe neurological abnormalities including convulsions in younger animals are ameliorated with age, and flailer homozygotes have a normal life span. Accumulation of SER in the Purkinje cell dendritic spines has been observed in older animals with the dilute-neurological class of mutations in Myo5a (unpublished data). Gradual accumulation of SER in the dendrites may occur in flailer homozygotes as well. Expression in the fully differentiated neuron of other components that function in SER transport and retention may also contribute to the improvement in older animals.
Interaction with the tumbler locus
The reason for the originally reported genetic interaction between tb and flr is not clear. One possibility is that the complementation studies were flawed. Only twelve animals from a single litter were scored in the original complementation studies, and the genetic integrity of this mating cannot be confirmed (H. Sweet, personal communication). The tb mutation is unfortunately extinct so the complementation experiment cannot be repeated. A more interesting explanation is that tb encodes one of the proteins besides MyoVA that is predicted to interact genetically with the flailer hybrid protein. One such potential class of proteins are the cargo-binding proteins that bind to the MyoVA tail. Future studies aimed at identifying and mapping MyoVA tail interacting proteins may help to resolve this question.
Exon shuffling and molecular evolution
The role of exon shuffling on an evolutionary time-scale is evident from the appearance of protein domains in novel combinations in many mammalian genes. One of the first recognized examples was the LDL receptor, which contains exons shared with at least five different genes (25). Another clear example is the combination of carbonic anhydrase and receptor tyrosine kinase domains in the CARP proteins (26). However, the events that bring diverse exons together in genomic DNA with in-frame coding potential are sufficiently rare that they have not previously been observed in human or mouse mutant alleles. The flailer mutant demonstrates that these rare events continue to occur in a complex genome, producing novel proteins which may subsequently acquire new functions and contribute to evolutionary innovation.
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MATERIALS AND METHODS |
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Animals.
B6CBACa-AWJ/A, tb2J/tb2J F2 mice were obtained from the frozen embryo repository at the Jackson Laboratory (Bar Harbor, ME). These mice were backcrossed once to strain C57BL/6J and heterozygous flr/+ offspring were intercrossed. flailer has subsequently been maintained by brother–sister mating of homozygous affected animals. The resulting recombinant congenic line, BcC-flr, is now at generation N10. The composition of BcC-flr is ~75% C57BL/6J and 25% CBA. C57BL/6J-d l20J/dv se mice from the colony at the NCI-Frederick Cancer Research and Development Center (Frederick, MD) (27) were crossed with flr homozygotes to generate the mice in Table 1. C57BL/6J and CAST/Ei mice were purchased from the Jackson Laboratory.
Motor coordination
. Mice were tested on a rotating rotorod drum (Ugo Basile Research, Milan, Italy). Rotation was accelerated from 4 to 40 r.p.m. during a 4 min trial. Four to six male mice of each genotype were tested at 5, 5.5 and 6 weeks of age. When placed on a narrow beam of 0.6 cM diameter, wild-type mice maintain their balance indefinitely whereas flailer homozygotes fall immediately.
PCR primers.
Primers for Myo5a were based on sequence from Genbank accession no. X57377:
M1R, nt 3890–3872 (5'-CCT TGG GTT GGA TGG CTT C-3');
M2R, nt 3793–3768 (5'-CTG TTC CAT GAG GAC TCG GTA AGC AG-3');
M3R, nt 3723–3700 (5'-GCT TTG CGC AAC TCA TTC AGC TCG-3');
M4F, nt 3409–3434 (5'-ATA CAC CTT CAG CTC TGA GTT TGC AG-3');
M5R, nt 3506–3481 (5'-GGA ATA GTG ACA TAT CCA GAG GCA CC-3').
Primers for Gnb5 were based on sequence from GenBank accession no. L34290:
G1F, nt 224–267 (5'-TGC AAA GAC AAG CGG AGA ATC GTG-3');
G2R, nt 318–295 (5'-GTT CGT AGT GAA GGA ATC CCA GAC-3');
G3F, nt 1104–1127 (5'-CGT TTC AAG AAT GTG TGT CCT CTC-3');
G4R, nt 1292–1269 (5'-GGC TTG AAG GTC CAG TTG AAG TGC-3').
Primer G5R (5'-TCA ATC ATT ACG CCC TGC TGG AGG-3') corresponds to nucleotides 421–398 of the Gnb5 intron (GenBank accession no. AF174491).
Genetic mapping.
The flailer mutation was mapped with a genome scan of phenotypic pools of 25–75 affected and unaffected F2 animals (28) using microsatellite markers (29) (Research Genetics, Huntsville, AL; list available by request). For RFLP analysis, the combined Myo5a cDNA probes SpecG (nt 1–1632) and D46-5' (nt 2904–3817) (GenBank accession no. X57377) detected a 3.6 kb TaqI fragment in strains C57BL/6J, C57BL/10J and CBA, and three unique fragments of 2.5, 1.8 and 1.7 kb in strain CAST/Ei. The Myo5a cDNA probe D46 (nt 2904–7156) detected TaqI fragments of 7.2 kb in strain CAST/Ei and 1.7 kb in strains C57BL/6J, C57BL/10J and CBA. Gnb5 was mapped on the Jackson Laboratory BSS backcross by single-strand conformation polymorphism analysis of a 166 bp fragment amplified from the 3'-untranslated region with primers G3F and G4R.
Northern blots.
Brain poly(A)+ RNA was prepared and analyzed as described (30). The 1.6 kb SpecG probe was used to detect transcripts containing the 5' portion of Myo5a. The 0.9 kb cDNA fragment D46-5' probe was used to detect transcripts containing the 3' portion of Myo5a. Gnb5 transcripts were detected with a cDNA fragment (nt 13–270, GenBank accession no. L34290) from EST no. 387224 (Research Genetics). Mouse alpha actin cDNA was used as a control for RNA loading.
5' RACE and RT–PCR.
5' RACE reactions were carried out with the 5' RACE System v2.0 (Life Technologies, Gibco BRL, Rockville, MD) using the three nested Myo5a-specific primers M1R, M2R and M3R with 0.2 µg of poly(A)+ brain RNA as template. For RT–PCR, first strand cDNA was synthesized by reverse transcription of 5 µg of total brain RNA using random hexamer primers and Superscript II reverse transcriptase (Life Technologies). The 5.4 kb Myo5a open reading frame was amplified from flailer brain RNA in 13 overlapping RT–PCR products and sequenced as described (4).
Genomic PCR and sequencing.
Genomic DNA was prepared from spleens of flailer mice. Wild-type DNA from strain C57BL/10J, the strain of origin of the flailer mutant, was purchased from the Jackson Laboratory. PCR was carried out with the Expand Long Template PCR System (Roche Molecular Biochemicals, Indianapolis, IN). The Gnb5 intron rearranged in flr mice was amplified using primers G1F and G2R. The Myo5a intron was amplified using primers M4F and M5R. The Gnb5/Myo5a hybrid intron was amplified with primers G1F and M5R. The Gnb5 intron probe used for Southern blotting was generated by PCR of genomic DNA using primers G1F and G5R. PCR products were gel purified and sequenced directly or cloned into the vector pGEMT. The University of Michigan DNA Sequencing Core (R. Lyons, Director) carried out the automated sequencing. Sequences were analyzed using Sequencer Software (Genecodes, Ann Arbor, MI).
Electron microscopy.
Mice were deeply anesthetized with Avertin and perfused transcardially with 4% paraformaldehyde/2% glutaraldehyde in phosphate-buffered saline (pH 7.2). Cerebellum was fixed in perfusion solution overnight and in 2% buffered glutaraldehyde for 1 h, post-fixed in buffered 1% osmium tetroxide for 1 h, dehydrated, and infiltrated with Epon epoxy resin. The block was sectioned and grids containing ‘ultra-thin’ sections were double stained with lead citrate and uranyl acetate. The Philips CM-100 transmission electron microscope was operated at 60 kV.
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ACKNOWLEDGEMENTS |
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We thank Scott Popma for assistance with the genome scan, Jennifer Kearney for advice on the rotorod test, Chris Edwards and the Cell Biology Laboratory (University of Michigan Medical School) for electron microscopy, Karl Herrup for unpublished light microscopy of flailer brain, and Sally Camper and David Ginsburg for helpful discussions. B.A.H. gratefully acknowledges space and support from Eric Lander during this work. This work was supported by NIH research grants GM 24872 to M.H.M. and TDK 25387 to M.S.M., and the National Cancer Institute, DHHS, under contract with ABL (N.A.J. and N.G.C.). B.A.H. is a Pew Scholar and recipient of a March of Dimes Basil O’Connor Starter Scholar Award, and was previously a fellow of the Helen Hay Whitney Foundation.
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FOOTNOTES |
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+ Present address: Department of Biochemistry, University of Hong Kong, 5 Sassoon Road, Hong Kong
§ To whom correspondence should be addressed. Tel: +1 734 763 5546; Fax: +1 734 763 9691; Email: meislerm@umich.edu
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